IT202000006031A1 - FLUID DEVICE FOR ANALYSIS OF BODIES AND RELATIVE METHOD - Google Patents

Description

DESCRIPTION of the patent for industrial invention having as title

FLUID DEVICE FOR ANALYSIS OF BODIES AND RELATIVE METHOD

The invention described here presents a device and relative method for measuring density. mass, weight, size and shape of corpuscles such as microspheres, cells, spheroids and organoids. In particular, the architecture of the device? suitable for the measurement of corpuscles having a size between one and five thousand micrometers.

The collection of detailed information on these parameters can? have an important impact in industrial and / or research applications in fields such as pharmacology, cell biology, agro-industry, food and environmental sectors. This growing need? attributable to the massive use of single cells and cellular aggregates on a wide range of biomedical applications.

In particular, the cells often show larger variations. accentuated on the density? mass rather than size, and obtaining reliable data on such values is a challenge to overcome. The determination of these values, can? thus providing an effective means of analysis for monitoring cellular responses to external stimuli, such as drugs and environmental changes. Furthermore, obtaining simultaneous information on the volume of the corpuscle and on the density? mass allows the correlation with the activities? biological, and obtaining important information. Among these are the level of organization and vitality? of the cell population within a cell aggregate.

STATE OF THE TECHNIQUE

The methods for determining the mass and density? of cells, corpuscles or molecules, have been implemented with the advent of nanoelectromechanical systems (NEMS), and in particular with the development of nano-mechanical resonators.

These systems are resonant when subjected to a specific frequency depending on their mass, structure and stiffness. The mass of the sample is measured by the variation in the oscillation frequency of the resonator when it interacts with it. The initial limit for the biological applications of this technique was due to the necessity? to operate in vacuum conditions. Suspended microchannel resonators (SMRs) have overcome this application obstacle, allowing you to measure density? of single cells in solution and thus obtain statistics on the density distribution.

However, using fluidic channels of micrometric dimensions, the systems mentioned above allow the analysis of corpuscles that are smaller than tens of micrometers. This ? an important technological limit for applications or studies on cellular aggregates such as spheroids and organoids that can reach diameters in the order of millimeters.

Are known methods to calculate the density? of phytoplankton and polystyrene spheres using the speed? of sedimentation. This technique does not allow to reach the average size of spheroids and organoids. Even more? important, the data is collected in a single step, without the possibility? to repeat the measurement for an output pi? accurate.

Other techniques employ an optical induced electrokinetics (OEK) system to lift the corpuscles previously introduced into a microfluidic channel.

Such a solution? expensive to make, and not? suitable for measuring corpuscles that have dimensions greater than twenty micrometers.

SUMMARY OF THE INVENTION

A first aspect of the present invention describes a fluidic device for measuring at least one of the densities. mass and weight of a particle. The fluidics device includes a sedimentation chamber fluidically connected to an inlet channel configured to be immersed in a liquid. The fluidic device further comprises a pumping system connected to the sedimentation chamber. The pumping system? suitable for controlling the flow of liquid in the sedimentation chamber. A fluidics device processor? configured to obtain particle data relating to a particle in at least one region of the settling chamber; and calculate at least one between density? mass and weight of the corpuscle based on the data received.

The liquid can? be a culture liquid or a solution, for example a saline solution, suitable for preserving and maintaining one or more? corpuscles or agglomerates of corpuscles to be introduced into the sedimentation chamber.

Thanks to the presence of an inlet channel directly connected to the sedimentation chamber,? Is it possible to make individual density measurements? mass and weight on a population of corpuscles. This configuration allows these measurements to be carried out in sequence and in an automated manner. The measured values, in combination with the particle data, such as the size and shape of the corpuscle, are important for example for the study of cell dynamics, or for the development of biological models of human organs.

In one embodiment of the first aspect, the fluidics device further comprises at least one sensing device configured to acquire the particle data and to provide the particle data to the processor.

In a further form of implementation, the fluidics device where the processor? configured to control the pumping system based on at least a portion of the received particle data.

In a further embodiment, the corpuscle data comprises at least one of the velocity, shape, position, size of the corpuscle.

In a further embodiment, the fluidics device further comprises a temperature control device, configured to provide the processor with a temperature measurement of a liquid in the settling chamber. The processor? configured to calculate at least one of the density? mass and the weight of the corpuscle also on the basis of said temperature measurement.

The temperature measurement allows to obtain precise measurements even in conditions in which the device is not in a controlled temperature environment. In fact, the motion of the corpuscle in the sedimentation chamber also depends on the viscosity? of the liquid in which it moves, which depends on the nature of the specific liquid used and its temperature.

In a further form of implementation, the temperature control device? also configured to regulate the temperature of the liquid in the sedimentation chamber based on at least one of the information provided by the processor and a predefined temperature value.

The temperature control allows to add to the measurement accuracy, also the maintenance of the ideal conditions for the maintenance of the corpuscle. For example, in the case where the corpuscle? consisting of biological material, the ideal conditions are to maintain it at 37 degrees centigrade, and in some protocols of use, the variation of the temperature may be required for the observation of specific biological phenomena.

In a further embodiment, the fluidic device further comprises a movable support suitable for housing at least a part of the at least one detection device. The processor? further configured to guide the mobile support based on at least a part of the received particle data.

Thanks to mobile support? Is it possible to follow the corpuscle during its movement, and therefore to obtain greater precision in the measurement of density? of mass and weight, and? It is also possible to carry out measurements on corpuscles that move at great speeds? in particularly long sedimentation chambers. Furthermore ? It is possible to obtain three-dimensional information on the corpuscle, thanks for example to the observation of the sample from different angles, or on different focal planes. The data obtained on the three-dimensional shape of the object allow a better reconstruction of the viscosity coefficient, and therefore a greater resolution of the density measurement. mass and weight.

In a further embodiment, the settling chamber further comprises a flow channel fluidically connected to the inlet channel and having an internal cross section that narrows into a portion of the flow channel.

In particular, the sedimentation chamber comprises a flow channel having at least two portions where the internal section of the first portion is larger than the internal section of the second portion.

The second portion? positioned in such a way that a corpuscle in the sedimentation chamber is able to move from the second section to the first section in the absence of flows in the liquid in which? immersed the corpuscle.

Variations in the section of the sedimentation chamber allow to obtain converging flow lines. During a phase in which a flow is produced in the sedimentation chamber, the corpuscle follows these lines of flow, and consequently moves towards a smaller region. central of the sedimentation chamber. Thanks to this phenomenon, measurement artifacts that depend on the interactions between the corpuscle and the lateral surfaces of the sedimentation chamber are avoided, and it is possible to produce more measurements. precise. This also guarantees the centering of the corpuscle even for long measurement times, thus enabling? the maintenance of the sample in the sedimentation chamber for biological experiments. The centering? also a useful aspect for the sampling, since? decreases the probability? to lose the sample in the fluidic system, for example by adhesion to the surfaces of channels.

In a further embodiment, the settling chamber comprises flow channels connected in parallel to each other and to the inlet channel. Each channel of flow? also connected to a flow regulator. In such an implementation the processor? configured to receive input data relating to a particle in the input channel, and control the flow regulators on the basis of said input data.

The presence of more? sedimentation chambers allows you to direct a corpuscle according to its size into a suitable sedimentation chamber. ? cos? It is possible to implement in a single instrument the measurement of corpuscles of even significantly different dimensions, for example from a micrometer to 5 millimeters. Alternatively, the presence of more? sedimentation chambers allows for a greater flow rate in the measurement of corpuscle populations.

In a further embodiment, the fluidic device further comprises a recirculation channel connected in parallel to the sedimentation chamber. Does the recirculation channel include a recirculation device, and the processor? configured to control recirculation device for the recirculation of the liquid in the sedimentation chamber.

Thanks to the recirculation system? possible to avoid the presence of more? of a corpuscle within the fluidic system and then carry out repeated non-destructive analyzes of individual corpuscles, and their recovery. In addition, the quantity? of analysis liquid used? less for prolonged measurements over time.

In a further embodiment, the fluidic device further comprises a secondary channel fluidically connected to the settling chamber. The processor? configured to selectively control the flow in the secondary channel to inject liquid into the sedimentation chamber and / or to expel liquid from the sedimentation chamber through the secondary channel.

The secondary channel can? be connected to the sedimentation chamber directly or through the inlet channel or through a secondary fluidic circuit.

The presence of at least one secondary channel allows the introduction of a new liquid into the sedimentation chamber during the measurement. ? cos? For example, it is possible to carry out biological experimentation protocols. Furthermore ? It is also possible to introduce new liquids suitable for the precision measurement of the specific corpuscle. For example, selecting from a panel of liquids to measure the liquid that has density? mass pi? close to that of the corpuscle,? measurement accuracy and accuracy can be improved. Furthermore, the same secondary channel can? be used for the removal of the sample and its sorting in specific containers. For example, through special fluidic derivations, different corpuscles can be collected in different containers, also based on the results of the measurements performed.

A second aspect of the present invention describes a method for measuring at least one of the densities? mass and weight of a particle. According to the method, a particle to be analyzed is introduced into a sedimentation chamber of a fluidic device through an inlet channel immersed in a liquid. Corpuscle data relating to a corpuscle in at least one region of the settling chamber are obtained. The corpuscle? moving in a liquid at rest in the sedimentation chamber. At least one between density? mass and weight of the corpuscle? calculated on the basis of the data received.

The measure can? be done while the corpuscle? moving in liquid at rest. By liquid at rest we mean that in the sedimentation channel not? there is no flow. In one implementation the corpuscle moves with accelerated motion subjected to a force field. For example, the sedimentation chamber can? be positioned in such a way that the corpuscle moves in the liquid at rest subjected to gravitational acceleration.

In one embodiment of the second aspect, obtaining the particle data comprises acquiring, by means of at least one sensing device, the particle data, and providing a processor with the acquired particle data.

In a further embodiment, the method further comprises controlling a flow in the settling chamber based on at least a portion of the received particle data.

In a further embodiment, the method further comprises measuring a temperature of the liquid in the settling chamber; and calculate at least one of the density? mass and the weight of the corpuscle also on the basis of said temperature measurement.

In a further embodiment, the method further comprises adjusting the temperature of the liquid in the settling chamber based on at least one of the information provided by the processor and a predefined temperature value.

In a further embodiment, the method further comprises guiding at least a part of the at least one sensing device based on at least a part of the received particle data.

In a further embodiment, the method further comprises recirculating the liquid in which? immersed the corpuscle to be analyzed in a recirculation channel fluidically connected in parallel to the sedimentation chamber.

In a further form of implementation, the method further comprises replacing, before and / or after a measurement of at least one of the density? mass and weight of the corpuscle, the liquid in which? immersed the corpuscle to be analyzed with a second liquid other than said liquid.

In a further form of implementation, the method further comprises selecting a particle based on one of the densities. of mass, weight, size and shape; and collect the selected corpuscle in a predefined container based on at least one of the densities? mass, weight, size and shape of the corpuscle.

The advantages described above in relation to the fluidic device also apply to the method described in relation to the second aspect and its implementation forms.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1. Front view of the sedimentation chamber containing the analysis medium and the corpuscle. Representation of the forces involved and the speed? terminal.

Figure 2. Schematic representation of the invention.

Figure 3. Schematic representation of the implementation related to the temperature control device.

Figure 4. Linear regression data obtained from the vertical variation of the position of the corpuscle as a function of time.

Figure 5. Experimental results of densities averages obtained on seven different PS microspheres measured for each lot analyzed (20, 50 and 90µm), and the respective standard deviations obtained on seven repetitions for each measurement.

Figure 6. A) Experimental results of the densities averages obtained on the three lots of PS microspheres, reported in comparison with the value declared by the manufacturer, and respective standard deviation. B) Speed terminal determined experimentally, in comparison with the speed? theoretical, for the three sizes of microspheres examined.

Figure 7. Schematic representation of the centering device (A). Enlargement of portion 100c (A, B) for the schematic representation of the centering of the corpuscle by pulsed flow (B) and by modulated flow (C).

Figure 8. Schematic representation of the sedimentation chamber comprising several flow channels having different sizes, connected to independent flow regulators, in order to satisfy the diversity? corpuscular.

Figure 9. Schematic representation of the technological solution relating to the measurement of the movement of large corpuscles, through the recognition of the passage of the corpuscle from different target positions at an adjustable and known distance.

Figure 10. Schematic representation of the dual camera system for bilateral morphological characterization of the corpuscle.

Figure 11. Schematic representation of the implementation relating to the acquisition of three-dimensional information on the corpuscle based on the acquisition of images reflected by more? corners.

Figure 12. Schematic representation of the implementation related to 3D image acquisition based on a sensing device orbiting with respect to a cylindrical sedimentation chamber.

Figure 13. Schematic representation of the implementation containing a recirculation channel, and relative secondary pumping system.

Figure 14. Schematic representation of the implementation containing a secondary channel for the introduction and exchange of further means of analysis.

Figure 15. Graphical representation of diameter and density monitoring of spheroids with respect to time, for the evaluation of their degree of maturation before treatment with the drug.

Figure 16. Schematic representation of the implementation dedicated to the Sorting of corpuscles.

DETAILED DESCRIPTION

? more and more? It is common for a single medical or biological laboratory to study corpuscles of different nature such as cells, microspheres, spheroids, organoids and / or other forms of corpuscular aggregates.

Given the huge variety? of samples and the interest in the measurement of weight, density, size and shape, the embodiments and implementations described below present technological solutions that allow to measure these parameters by adopting a single device and various methods of use associated with it. An embodiment of the present invention describes a first solution which allows such measurements to be carried out. In one example, the measurement of weight, density, size and shape can be carried out concomitantly, simultaneously or in combination on samples having dimensions ranging from one to five thousand micrometers. In addition to having the advantage of operating with a single instrument for a large dimensional distribution of samples, this? allows a solid correlation between the data obtained on the individual corpuscles.

The embodiments of the fluidic device and related methods described in the present disclosure allow to carry out the analysis described above in an economical, accurate and non-invasive way. Such fluidic methods and devices can be used to perform measurements not only on organic and biological targets, but also on inorganic samples.

The following paragraphs describe several exemplary embodiments of the present invention. By way of example, and to facilitate understanding, the embodiments will be described with reference to the analysis of biological material such as cells or cellular agglomerates, hereinafter referred to by the generic term of corpuscles. It is however understood that the method and the device relating to the invention presented here allow to measure density? mass, weight, size and shape of non-biological corpuscles. In particular, the implementations with reference to the exemplary embodiments described below can also be used to analyze corpuscles other than cells or cell agglomerates. Consequently, the term "corpuscle", used in the context of the embodiments of the present invention to indicate cells, cell agglomerates or in general biological material can be used. also be used to indicate microparticles, residual particles from industrial production processes, airborne airborne dust, pollen, vesicles, oil droplets in aqueous suspension, air bubbles in liquids.

Similarly, the term "means of analysis" or the term pi? generic? liquid? ? used in the explanatory examples of the present invention to indicate a medium compatible with cell cultures. However, it will be? it is clear to one skilled in the art that the term? means of analysis? and the term? liquid? must be interpreted in their sense pi? general. In particular, based on the design needs and the nature of the corpuscles to be analyzed, sar? clear to the skilled in the art that the liquid can? be a cell culture medium or other liquids, such as aqueous-based solutions, or oils.

The present invention has the purpose of bridging the gap within the state of the art concerning the possibility? to use a single technological apparatus, and the relative method of use, for the concomitant measurement of density? mass, weight, size and / or shape of corpuscles also of different sizes. In fact, the devices present today can operate partially in a narrow dimensional distribution. Furthermore, they do not allow to obtain simultaneous or combined measurements of these parameters. There? leads to the obtaining of fragmented information, difficult to correlate with each other.

Even if the object? been described with particular reference to the attached figures, the reference numbers used in the description and in the claims are used to improve the intelligence of the invention and do not constitute any limitation to the scope of protection claimed.

The invention presents a new device, and the relative method of use, capable of carrying out the concomitant measurement of density. mass, weight, size and / or shape of biological and non-biological corpuscles such as microspheres, cells, spheroids and organoids, having a size between 1 and 5,000 micrometers.

After a detailed description of the various implementations suitable for the realization of this measure, multiple modalities will be disclosed? of use of the present invention. The latter include readjustments of the basic system, with particular reference to their usefulness. in the biomedical field. The present invention? suitable to analyze a corpuscle (420) in motion in conditions of flow at rest, guided by the force of gravity? (Figure 1). Such a movement of the corpuscle? mainly influenced by the density? of mass, from the volume, from the shape, from the orientation of the same and from the density? mass and viscosity? of the surrounding liquid.

A schematic representation of an embodiment of the present invention? shown in Figure 2.

The embodiments described with reference to Figures 3 to 16 describe subsequent improvements and developments of the fluidic device of Figure 1. Individual features described below with reference to a single figure can therefore be combined with features described with reference to the other figures.

The fluidic device comprises a sedimentation chamber 100, a pumping system 200, a detection device 300 and a processor 500. The movement of the corpuscle 420 occurs inside a fluidic channel with transparent walls, here called the sedimentation chamber 100. The pumping system 200 includes a flow generation system 230 which, by way of example, can? be a peristaltic pump or a pressure control system. An inlet channel 210 and an outlet channel 220 can be connected to a loading or unloading tank, alternatively they can be used as a vent. The system comprising the detection device 300 connected to the processor 500 performs the dual role of monitoring the movement of a corpuscle 420, and of measuring its shape and size. Both the sedimentation chamber 100 and the sensing device 300 are sized to allow accurate measurements of the movement, shape and size of the corpuscle 420, as described in detail later in the text.

Although the device of Figure 2 includes the sensing device 300, the sensing device? optional. in alternative implementations, the fluidic device pu? be made without the detection device.

In particular, in the context of the embodiments of the present invention, by "measuring the shape and size" of the corpuscle is meant the measurement of any characteristic of the corpuscle that can be used to represent or approximate the three-dimensional geometry of the corpuscle. This could coincide with the measurement of the radius, in the case of a spherical corpuscle, or with other geometric characteristics in the case of nearly spherical and non-spherical corpuscles.

For a correct functioning of the device, the corpuscle 420, contained inside the analysis means 450, is taken from the tank 400, introduced and transported, inside the sedimentation chamber 100 through the inlet channel 210. The transport of the corpuscle inside the pumping system 200 occurs through the actuation of a system for the generation of flow 230. After the introduction of the corpuscle 420, the flow is blocked and the corpuscle moves inside the analysis medium 450 driven by the force of gravity. In the absence of other flows, and after a transient time due to an acceleration phase, the corpuscle will reach? its speed? constant, here defined speed? terminal, speed? of drift or speed? of sedimentation.

Once the measurement has been carried out, the input channel 210 and the output channel 220 can be used indifferently for the recovery of the corpuscle 420.

The device can? be used to make relative measurements, in which the differences between different corpuscles are evaluated, or to make absolute measurements. In the last case,? useful knowledge of the temperature, in order to define the density? mass and viscosity? analysis means 450. In one embodiment of the present invention the device? adapted to be used inside a controlled temperature environment, such as in an incubator. Alternatively, the temperature can? be measured during the experiments. In an embodiment (Figure 3) of the present invention, the average temperature of the analysis medium 450 is measured by means of a temperature control device 600 composed of at least one sensor 610, positioned in the vicinity of the sensor. of the sedimentation chamber 100. Then the processor 500 adapts the viscosity values? calculated as a function of the measured temperature.

In another embodiment, when operating conditions require a specific temperature value (e.g., 37 ° C for live biological samples), the temperature controller 600? configured to adjust the liquid temperature in the sedimentation chamber based on information provided by the processor 500, such as the current temperature value, particle type, viscosity? of the liquid, the current value of the viscosity? liquid, etc. In a specific embodiment, the temperature control device 600 manages the unit? temperature regulation 620, which allows the maintenance of a specific value during the analysis. For example, the temperature controller 600 can? comprise several heaters (620a and 620b) and sensors (610a and 610b), located in various positions of the fluidic device, of the system of pumping system 200, and in particular in the vicinity? of the sedimentation chamber 100.

From the physical point of view, and in order to maximize the accuracy of the data obtained, the movement of the corpuscle can? be considered when reaching its speed? terminal, and not during the transitional period. The speed? relative of the corpuscle with respect to the means of analysis v (t), pu? be obtained by solving the dynamic equation in the equation of motion:

Where is it ? ? the transitional period of the dynamics of the corpuscle:

where mp? the mass of the corpuscle,? p? the density? mass of the corpuscle, at? the acceleration of the corpuscle,? m? ? the density? mass of the analytical medium, Vp? the volume of the corpuscle, see? the speed? of drift of the corpuscle, v0? the speed? initial of the corpuscle, r? the radius of the corpuscle, k? the coefficient of friction ,? ? the viscosity? of the analysis medium and g? gravitational acceleration.

In order to have an estimate of the order of magnitude of this transitional period, the following calculations are reported in the case of biological corpuscles of different sizes and types, measured in aqueous solutions. Do water-based solutions have a viscosity? about 1 mPa? s, and the density? mass of biological corpuscles? on average 1020 sheets /? m3. In the case of corpuscles with a diameter between 1 and 2,000 micrometers, the order of magnitude of? varies from 60 ns (for a diameter of 1? m) to 250 ms (for a diameter of 2,000? m). In this interval, the corpuscles reach the speed? terminal in a short time. Since? this time frame? negligible, the acceleration of the corpuscle has no impact on the measurement and the dynamics of the corpuscle pu? therefore be considered as a uniform linear motion.

Other technical implementations of the embodiments of the present invention allow the measurement of larger corpuscles, in particular between 2 and 5 millimeters in diameter. For such implementations the transient time must be considered, as it can? reach 1.5 seconds in the case of samples of 5 millimeters in diameter.

For example, in an embodiment of the present invention, the corpuscle is brought to a predetermined distance above the working region of the detection device 300, in order to exclude the transitional period from the measurement.

In another implementation of the present invention, the working region of the sensing device 300? sufficiently large, and the settling chamber 100? long enough to allow the detection of the complete dynamic movement of the corpuscle, including acceleration and velocity? terminal. The data collected during the transitional period of the corpuscular motion may not be considered for the calculation of the velocity? terminal.

In an embodiment of the present invention, the theory used for the measurement is based on an elaboration of Stokes' law, in which:

(eq. 1)

And

Where Wp? the weight of the corpuscle.

In order to demonstrate the reliability? of this invention, a specific implementation of the same (Figure 2)? was used for the measurement of the density? mass and size of polystyrene microspheres between 20 and 90 micrometers.

In such an implementation, the detection system 300? composed of an optical microscopy system with 4X magnification. The sedimentation chamber 100? obtained inside a transparent chip of polydimethylsiloxane (PDMS) adhered covalently to a slide. The architecture of the sedimentation chamber 100 has a length of 6 cm, and a cross section of 1 x 1 mm. The corpuscle is positioned in the upper part of the sedimentation chamber 100 by a flow generation system 230, constituted in this implementation by a peristaltic pump. Prior to its arrival in the sedimentation chamber 100, the sample is transported through the pumping system 200, made up in this case of polytetrafluoroethylene (PTFE) tubes. the processor 500? composed of a computer and relative software, capable of processing the images collected by the unit? of microscopy. From these images are extrapolated the two-dimensional projection of the shape of the corpuscle and its speed? terminal. The latter is calculated through linear regression obtained from the variation of the position of the corpuscle's centroid as a function of time (Figure 4). the processor 500 then calculates the density? mass and the diameter of the corpuscles by adopting the mathematical model previously presented. This procedure is repeated more? times on each particle to obtain statistically significant results.

This specific implementation? was used to measure corpuscles with density? and known diameters for the purpose of validating the invention. In particular, three batches of polystyrene (PS) microspheres (Polybead ?, Polysciences Inc. US) having a diameter of 20, 50 and 90 micrometers were selected. The product sheet provided by the seller indicates, for the PS spheres, the value of the density? of average mass equal to 1,050? 10 sheets / um3. Seven units were analyzed for each batch of microspheres. different, and the size? was repeated seven times for each unit, in order to increase the significance? statistics. Figure 5 shows the density value? mean of the seven different microspheres analyzed, and the respective standard deviation extrapolated from the seven repeats for each lot. In line with the experimental expectations, the standard deviations of the measurements performed on all the microspheres are comparable, demonstrating the accuracy and reliability. of this implementation.

Furthermore, the values of density? obtained for the PS microspheres, were found to be considerably more? accurate to the average value declared by the manufacturer (Figure 3a). In particular, it can? note that the average standard deviation turns out to be an order of magnitude less than that of the commercial value (Figure 6a). A further confirmation of the accuracy of the results is obtained by comparing the speed? theoretical terminal (eq.1) and the experimental one (Figure 6b).

Specifically, the same configuration presented in Figure 2, used for the validation analysis carried out on the PS microspheres, can? be adopted for the analysis of large biological samples, such as spheroids or organoids.

The relevance of such applications concerns the possibility? to use the method presented here for example to monitor the growth phases of the spheroids. It is in fact an important aspect that allows us to understand the moment in which a population of spheroids reaches maturity? necessary, or desired by the operator, to continue the biological experimentation. The process of formation of the spheroids in fact undergoes a compaction over time, starting from an agglomeration of disintegrated cells, and reaching a compact aggregate stage. There? ? particularly important in biomedical fields, where the biological material normally used for analyzes is not? standardized, bringing cos? to obtaining less reliable results. The simultaneous measurement of density? mass, together with the weight, shape and size of the sample, could allow us to understand the best conditions for the growth of standard spheroids. Furthermore, the spheroids can be generated from different types of cells or from a combination of them, consequently both the time it takes to reach maturation and the density? of mass characteristic of the mature phase, they are different for each type of spheroid. An important application field for which? important to determine the value of the density? mass during the spheroid formation process, relates to the industrial drug screening process. In this context, can it? the efficacy of a pharmacological treatment must be assessed by studying the absorption of the drug in the spheroids themselves. ? in fact known that the penetration of the drug? highly correlated to the level of compactness of the aggregate, which in turn can? be put in relation with its density? mass. Therefore, the evaluation of the correct structure of the spheroids, in terms of density? mass, can? greatly improve the reproducibility? of the results. This implementation of the invention aims to improve this aspect of the research which currently represents one of the main limits for the application of 3D biological models, aimed at the discovery and development of drugs.

However, spheroids are not the only corpuscles used in the biomedical field. The various nature of the corpuscles is also reflected in the vast range of dimensions they cover, ranging from single cells to organoids. Known the interest of the researchers on the whole range of corpuscles, the possibility? to carry out the simultaneous measurement of their density? mass, weight, size and shape, through a single tool, would be of considerable impact for the community? scientific.

In this regard, the following paragraphs describe the methodological and / or technological implementations adopted to expand the dimensional range of the corpuscles that can be analyzed.

For example, in an adaptation of the invention, for the analysis of micrometric corpuscles? a high-magnification optic is required, capable of having a resolution sufficient to detect its size and shape. Another adaptation concerns the fact that, generally, a particle of millimeter size tender? to have a speed? of fall pi? high compared to a micrometric-sized corpuscle. Consequently, the settling chamber 100 must be long enough to allow its velocity to be measured. terminal. Therefore, the correct selection of a specific detection device 300 and of the sedimentation chamber 100 could be relevant to broaden the operative field of the invention.

In an example of the previously described implementation, the sensing device 300? composed of an image acquisition system, which acquires a series of frames of the corpuscle 420 during its movement inside the sedimentation chamber 100. These images are processed to obtain the size, shape and position of the centroid of the corpuscle with respect to time. The speed? terminal is calculated using a linear regression algorithm, as shown in Figure 2. This specific implementation requires that a sufficient number of images of the moving corpuscle are collected in order to obtain a correct number of points, which will then be used by the regression. For example, in conditions of spherical corpuscles, an acceptable number of points could be 5, while in conditions of non-spherical corpuscles, the acceptable number of points could be more? high due to the additional uncertainty in recognizing the centroid of the corpuscle. Specifically, the number of points that the device? able to obtain during a measurement depends on several experimental and technological factors. These include speed terminal of the corpuscle, the length of the portion of the sedimentation chamber observable by the recognition system, the frame rate and the optical resolution. These factors can be combined in a formula that predicts the number of analysis points detected in the specific technological and experimental conditions, reported here as:

In a particular implementation of the embodiment of the present invention, the acceptable number of analysis points? set to a specific value, for example 10. Otherwise, in another embodiment of the present invention, such an acceptable number of analysis points? variable, and? regulated by an optimization algorithm. For example, the algorithm can? adjust the ideal number of analysis points based on the quality? the fitting of the position data of the corpuscle, as a function of time.

Furthermore, in order to improve the accuracy of the measurements, the device allows to position the corpuscle in the center of the sedimentation chamber before carrying out the analysis. For example, in an embodiment of the present invention, the device uses a system for centering the corpuscle by means of a variation of the geometry and / or a narrowing of the sedimentation chamber 100. The operating principle is based on the use of a specific geometry. of the sedimentation chamber which allows to obtain laminar flows inside it with a non-zero horizontal component. In such conditions, through an appropriate control of the pumping system 200, the corpuscle can? be transported to the area of the sedimentation channel where the flow has a zero horizontal component. Below, and more? in general, in the context of the present invention, vertical means the direction parallel to the gravitational force vector, and horizontal means any vector perpendicular to the vertical direction.

For example, in the case in which the sedimentation chamber is arranged vertically, if the corpuscle is in a position, inside the sedimentation chamber, where the flow lines have a non-zero horizontal component, the corpuscle is also affected by a thrust. lateral due to the horizontal component of the flow. In a particular embodiment of the present invention, the sedimentation chamber 100,? vertically oriented and? consisting of two portions having a horizontal section of different sizes, so that the section channel pi? wide (100a) is located below the channel with the pi? narrow (100b), as shown in Figure 7A. Furthermore, in this particular example, the two portions (100a and 100b) of the sedimentation chamber (100) are coaxially connected by a portion of the channel with an inclined section (100c). The time required for the centering of the corpuscle can? vary, according to the value of the angle of inclination of the walls of the portion 100c, of the percentage of narrowing of the section of the portion of the sedimentation channel pi? small (100b) compared to the pi? large (100a), or the ratio between the areas of the two sections (Figure 7A). In any case, the specific value of these parameters is not? essential for the operation of the centering system. In a further embodiment of the present invention, the inclined section channel portion (100c)? replaced by a discontinuous change of section, or by walls at 90? with respect to the vertical axis.

A specific example of the centering system? presented in Figure 7A in order to describe its operation. In this particular example, the geometry of the pi? of the sedimentation chamber 100 has a square section having a side 2 mm (100a), while the larger portion? of the sedimentation chamber 100 has a square section having a side of 1 mm (100b). The two portions are connected by a narrowing portion (100c), in this case gradual, having an angle of 45? with respect to the longitudinal axis of the sedimentation chamber. The measurements concerning the square section of the sedimentation chamber given in this example are indicative. The relationship between the sections of the two portions of the sedimentation chamber and the inclination of the narrowing portion are therefore not limited to those deriving from these measurements. The dimensions given in this example are indicative and these particular values are optional.

In a particular method of using the centering device, the fluidic system is regulated in a pulsed manner. In one example the corpuscle 420 is in the position described in Figure 7B, at time t1 (420t1). Once the pumping system 200 has been activated, the corpuscle is transported following the flow lines imposed by the geometry, until it is positioned as shown in Figure 7B at time t2 (420t2). The flow is then interrupted, allowing the corpuscle to begin its fall under the balanced action of the gravitational and hydrostatic force. Following this, the corpuscle will be found? in a position pi? centered with respect to the sedimentation chamber 100, at time t3 (420t3). The further activation of the pumping system 200 allows the repetition of the process until the centering of the corpuscle 420 is reached.

In a further method of using the centering system, the corpuscle 420 is maintained in the narrowing portion 100c by means of a specially modulated flow inside the sedimentation chamber 100 in order to balance the gravitational action, as indicated in Figure 7C. In this way, the lateral component due to the flow lines generated by the variation in the geometry of the channel acts as a lateral thrust, moving the corpuscle from a lateral position at the time ta (420ta) to a position pi? central at time tb (420tb).

For both solutions presented, the pumping system 200 pu? be operated manually or automatically.

In one embodiment of the present invention, the sensing device 300? consisting of an optical device comprising a microscopy system mounted on a mobile support. The resolution of the optical device? able to acquire images of all the corpuscles of interest, reaching a level of detail sufficient to process their size and shape. Mobile support? driven by the processor 500 to follow the movement of the corpuscle within the settling chamber 100, so that the observed drop length may be long enough to measure enough points to achieve reliable speed processing. terminal of the corpuscle 420. Likewise, in another embodiment, the movable support? adapted to move the sedimentation chamber 100, so that the corpuscle 420 remains within the working range of the detection device 300.

In another embodiment, the detection device 300 comprises an optical system capable of supporting various magnifications. This is achieved, for example, by means of a mechanically operated optical axis. The processor 500 selects the correct magnification for the specific corpuscle under examination, and what is more? able to select the right balance between resolution, for the measurement of the properties? geometry of the corpuscle, and size of the visual field. This last one will have to be large enough to allow the processing of the speed? terminal of the corpuscle.

In another embodiment,? You can select the correct magnification to measure a specific particle without the aid of moving parts. In this embodiment, the settling chamber comprises several flow channels and the detection device monitors the different flow channels, each with a different magnification. Before the measurement, the detection device recognizes the corpuscle and the processor manages its introduction into the appropriate flow channel. Furthermore, the architecture of the flow channels can? be made in different configurations, in order to meet the need for diversity? corpuscular. In fact, dimensions, cross sections and geometries can be varied, with particular attention to the length of the channel. In this way, a larger corpuscle is inserted into a smaller canal. long and is monitored at a lower resolution for a longer field of view? ample. Otherwise, a corpuscle pi? small is inserted in a channel pi? short and is monitored at a resolution pi? high for a field of view more? small.

In a variant of this particular embodiment, the mechanism for introducing the corpuscle into the sedimentation chamber 100? a fluidic multiplexer. In a further variant of this embodiment (Figure 8), the sedimentation chamber comprises several flow channels (for example 110a, 110b and 110c) connected to independent flow regulators (for example the opening / closing valves 271a, 271b, 271c). All flow channels are connected in parallel within the fluidics device. In this case, the sensing device 300 recognizes the particle before it reaches the branch, in a specific optically accessible channel 150, and selects the flow channel in which the particle will come. introduced through the control on the valves. In another variant of this embodiment, the operator manually selects the flow channel used for the measurement, operating on a selection valve positioned on the inlet channel 210.

A different strategy to allow the measurement of the movement of particularly large corpuscles consists in a different method of localization of the same, in which the sensing device 300? capable of recognizing the passage of the corpuscle 420 through different target positions in the sedimentation chamber 100. The distance between these target positions, as well as the overall distance covered by this localization system, can? be adjusted in order to precisely determine the speed? terminal of the corpuscle. The speed? terminal of the corpuscle? then determined by dividing the distance traveled by the elapsed time.

In a technical implementation of this device, and its method, the detection device 300 comprises several image recognition systems dedicated to the target positions of the sedimentation chamber 100. The latter, in addition to contributing to the measurement of velocity terminal, they collect information on the shape and size of the corpuscle. In another technical implementation, the sensing device 300 comprises a series of sensors (e.g. 315a, 315b and 315c in Figure 9), which are located at the target positions (e.g. 115a, 115b and 115c) of the settling chamber 100 , and an independent image recognition system 320 for collecting the shape and size information of the corpuscle 420.

A further strategy to allow the measurement of the movement of particularly large corpuscles consists in acting on the density? mass and viscosity? of the means of analysis. For example, in one embodiment of the present invention, the? adapted to host an analysis medium 450, with a density? of specific mass for the type of corpuscles analyzed 420, and in particular corresponding to the density? average mass of the corpuscle population (for example 1035 fg / um3 in the case of SW620 spheroids). So, the total force acting on the corpuscle, or the sum of the gravitational and hydrostatic forces,? directed downwards or upwards depending on the density? mass of the corpuscle. Consequently, during the measurement phase in the absence of flow, the corpuscles pi? dense of the analysis medium will tend to move downwards, vice versa for the less dense ones.

In this specific embodiment, the processor 500 and its processing algorithm are adapted to calculate the speed. descending or ascending terminal. In addition, the system for generating flux 230? capable of promoting a flow inside the sedimentation chamber 100 both for and against the gravitational vector.

This allows you to move the sample in the opposite direction to its velocity. terminal, thus allowing the measurement to be repeated. Likewise, in another embodiment of the present invention, the? adapted to house an analysis medium 450 with high viscosity, which decreases the speed? terminal of the corpuscle.

In another embodiment of the present invention, the device? adapted to change the analysis medium 450, with one or more? analysis means 450 having density? of different mass. The device architecture of this implementation? the same architecture described in Figure 2, in which the pumping system 200? adapted to select the new analysis medium to be introduced into the system. In this case, the sensing device 300 measures the speed? terminal of the corpuscle, and activates the pumping system 300 to modify the analysis medium in such a way that the velocity? terminal corpuscle decrease. This protocol can be repeated until the speed? terminal of the corpuscle is low enough to be easily detectable and measurable.

In the foregoing description of the embodiments of the present invention and of some of its implementations, methods and related tools for carrying out combined density measurements have been described. mass, weight, size and / or shape of a large set of spherical corpuscles. However, many of the biological samples used in biomedical fields, including the aforementioned samples, may exhibit some deviation from sphericity. In particular for spheroids and organoids, the sphericity? depends mainly on the variability? local of the composition, and / or of the activity? cellular during the formation and maturation of the aggregates themselves. Individual cells can have slight variations from the spherical shape, due, for example, to the internal architecture of the cytoskeleton, to an uneven external pressure of the extracellular matrix or to the presence of rigid cell structures. Therefore, in the following paragraphs variations of the present invention are disclosed which take into account the cases in which the three-dimensional shape of the corpuscles deviates from the sphericity.

Unlike the case of a spherical corpuscle, for which many detection methods and algorithms are available to calculate the coefficient of friction in relation to its radius, the case of a non-spherical corpuscle appears to be more? complex, and requires the generalization of the previously discussed theory.

In particular, equations 1 and 2 generalize to the following:

And

In this form, both the friction coefficient k and the volume Vp depend on the shape and orientation of the corpuscle. There is a vast literature on theories that elaborate the geometric characteristics of an object to approximate the coefficient of friction k. These geometric characteristics can be ellipticity, or the ratio between the surface area of a sphere having the same volume as the corpuscle under examination and the real surface of the corpuscle itself. Furthermore, the coefficient of friction k pu? theoretically be calculated through computational fluid dynamics simulations, if the complete 3D shape of the corpuscle? Note.

Therefore, the term "spoke" used in the context of the embodiments of the present invention, including the preceding paragraphs,? intended as a geometric radius in the case of a spherical corpuscle. However, the term radius? to be understood as? effective ray ?, and can? also be used to mean a one-dimensional geometric feature representing a nearly spherical or non-spherical corpuscle. For example, the term radius pu? be understood as the average distance of the surface points from the centroid, or even as twice the ratio of the two-dimensional area to the perimeter, or as the Feret radius (perimeter divided twice as pi).

Consequently, as previously written, the terms "shape" and "size" used in the context of the embodiments of the present invention are intended as any geometric description of the corpuscle, which can be used to represent or approximate its two-dimensional or three-dimensional geometry. For example, it can? be understood as a measure of the radius, in the case of a spherical corpuscle, or a measure of other geometric characteristics in the case of nearly spherical and non-spherical corpuscles.

Also, accordingly, the term "mass density" or simply "density", used in the context of the embodiments of the present invention, is. understood as the ratio between mass and volume in the case of a spherical corpuscle. However, these terms can also be used to mean "effective mass density", which includes beyond the definition of density. previously described, even variations dependent on form factors, such as ellipticity? and roughness? superficial in the case of nearly spherical and non-spherical particles.

Likewise, the term "weight" used in the context of the embodiments of the present invention? understood as gravitational mass in the case of a spherical corpuscle. ? instead intended as "effective weight" in the case of non-spherical or nearly spherical corpuscles. Similarly to what is described for the term "density of mass", the term "effective weight" includes variations that depend on form factors such as ellipticity. and roughness? superficial.

As previously described in different embodiments of the present invention, the execution of different measurements of the same corpuscle allows to increase the reliability. of the results. In the case of spherical corpuscles, the statistical distribution obtained? correlated exclusively to the uncertainty of measurement. Differently, for quasi-spherical and non-spherical corpuscles, the experimental distribution of the measurement of velocity? terminal, can? be used to describe the size and shape of a corpuscle. In a variant of this embodiment, the standard deviation of the velocity? terminal can be used as a parameter to determine the ellipticity? corpuscular. This information ? relevant, for example, for the generation and selection of uniform spheroids, to be used as a model for drug development. In the different variants of this embodiment, the asymmetry of the distribution of the data obtained from the measurements can? be correlated to geometric factors of symmetry, the kurtosis index can? be related to heterogeneity? of the form, and the multimodality? can be related to the distribution of the two-dimensional projections of the corpuscle.

In a further implementation of the embodiments of the present invention, the images of the corpuscle are processed to obtain the three-dimensional reconstruction thereof, for example, through the use of tomographic algorithms. This reconstruction can? therefore be processed by computational tools for the calculation of the friction coefficient. This approach improves the measurement of the absolute value of the density? mass for nearly spherical and non-spherical corpuscles.

In particular, different technological implementations can be adopted to obtain the reconstruction of the 3D shape of the corpuscle or to improve its morphological characterization.

In a specific implementation of the embodiment of the present invention, the sensing device 300 includes a dual camera system (Figure 10). The orientation of the cameras, perpendicular to each other with respect to the sedimentation chamber 100, guarantees the acquisition of the images of the corpuscle from two different angles. These images are then processed by the processor 500, to obtain the 3D model.

Otherwise, in the case of using a single image acquisition system,? It is possible to adopt different optical, fluidic or mechanical strategies. For example, in another implementation of the embodiments of the present invention, the fluidic device includes within it two optical mirrors 113 and 114, in proximity to it. of the rear walls of the sedimentation chamber 100, and having a predetermined inclination with respect to the detection device 300 (Figure 11). The front camera maintains the focal plane such as to include the reflected images of the corpuscle 430 and 440 obtained respectively by the mirrors 113 and 114. By means of this implementation,? It is possible to simultaneously collect images from different spatial perspectives of the corpuscle and use them for the 3D reconstruction of the same.

In another implementation of the embodiments of the present invention, the device? adapted to acquire image stacks of the corpuscle 420. In such an implementation the sensing device 300? composed of at least one camera, in which the plane of focus is adjusted to allow scanning of the corpuscle by acquiring multiple images. Such collected images can be processed by the processor 500 through the application of image filters such as deconvolution, to carry out the 3D reconstruction of the corpuscle. Further embodiments of 3D corpuscle reconstruction, such as other image stacking techniques, holographic microscopy, or light-sheet microscopy, can be implemented.

In a further embodiment of the present invention, the sensing device 300? composed of an imaging system that orbits the settling chamber 100, as shown in Figure 12. In a specific implementation of this embodiment, the settling chamber 100? cylindrical. There? it allows to acquire images from different angles of the corpuscle which are then processed, through a tomographic reconstruction, for the 3D reconstruction of its morphology. In this specific implementation, are reference marks used in proximity? of the sedimentation chamber 100 to decrease the uncertainty in the measurement of the position of the corpuscle 420 introduced by the rotation of the detection device 300. The same strategy can? be used to minimize the uncertainties introduced in any other implementation that includes moving parts of the sensing system 300. In a variant of this embodiment, the rotating sensing device 300? adapted to move vertically and follow the movement of the sample in correlation with its velocity? terminal. The above implementations and methods adopt known technologies, rearranged to obtain the 3D reconstruction of the corpuscle. There? nevertheless, other solutions existing but not reported in this document can be combined with the embodiments of the present invention.

The previous paragraphs have described the method, the technology and the modality? of main use of the invention. However, different modalities? of use of the invention will be reported herein in order to demonstrate further advantages thereof.

For example, some alternatives may concern: combined measurements of corpuscle populations, imported for high throughput analyzes (increase in the volume of data produced); culturing and prolonged analysis of the corpuscle over time, relevant for applications such as drug testing; and corpuscle sorting, relevant for medical applications or for standardization of biological models.

High throughput analyzes, obtained from studies performed on a large number of single corpuscles, or on populations, are extremely important in biological fields. For example, information such as heterogeneity? of the population under examination, allow to understand the specific behavior of the subpopulations present within the biological sample. In fact, this behavior often tends to have a notable variation from the average of the entire population, and becomes an important discriminatory analysis factor. Nowadays, the instruments for the measurement of density? mass, weight, size and shape of each corpuscle of the population are not available on the market.

The invention proposes the solution to this gap in the state of the art, also allowing the extrapolation of the data necessary for the statistical analysis of the corpuscular population in general through an implementation of the fluidic device where the particle data that the processor 500 receives from the detection system 300 are related to a multitude of corpuscles, and include at least one of speed, shape, position, and size of pi? of a corpuscle. A specific embodiment exploits the same scheme described in Figure 2, where detection system 300? composed of a microscopy system, connected to the processor 500. This implementation allows the concomitant measurement of density? mass, weight, size and shape of each particle of the population. Similarly to the main mode of use described above, the particles 420 contained in the analysis medium 450 are collected from the reservoir 400 and introduced through the inlet channel 210 of the pumping system 200. The flow generation system 230 is activated to guide the corpuscles into the sedimentation chamber 100, the flow is subsequently stopped and the analysis is carried out during the fall of the corpuscles by gravity. In this specific mode? of use, the sensing device 300? adapted to simultaneously monitor each particle 420 present inside the sedimentation chamber 100 during the measurement. The speed? terminal and the size of each individual component of the population, are calculated by indexing each corpuscle 420 monitored inside the sedimentation chamber 100. In this way, the distributions of density? mass, weight, size and shape of the population are extrapolated after several repetitions of the measurement, while the information on each particle is still maintained.

In another implementation, a larger volume of data? obtained through particular geometries of the sedimentation chamber 100. Although in the devices illustrated in the figures the sedimentation chamber 200 or the flow channels 110 included therein are straight and do not have curves, the sedimentation chamber 200 can? alternatively have a geometry that is also curvilinear. In this embodiment, the settling chamber can? for example comprising a single channel having a serpentine architecture, so that several of its vertical portions, in which the analysis takes place, can be monitored by the detection device 300. The high throughput, applied to the embodiments of the present invention, allows to extrapolate important data such as the statistical distribution of the corpuscular population analyzed. For example, a normal (or Gaussian) distribution provides information on homogeneity? of the population. The analysis of the standard deviation of this distribution, can? therefore be relevant to define the quality? of a sample. This ? important for example in the case of cellular databases, which can certify the quality? of available samples. Otherwise, a bimodal or multimodal distribution can? identify the presence of different subpopulations or categories. For example, it can? provide information on the various cell lines present in a heterogeneous population, or can it? distinguish the cells that are in different phases of the cellular life cycle, in the case of homogeneous populations. Furthermore, still considering the case of a homogeneous population subjected to pharmacological treatment, a difference in the statistical distribution from normal to bi- or multi-modal, can? discriminate for example the permeation of the drug and / or its effectiveness.

Another aspect of the invention aims at avoiding the waste of the analysis medium, by means of a device capable of keeping the corpuscle suspended inside the sedimentation chamber, through a closed-loop fluidic system. There? ? particularly important for applications that use expensive means of analysis, such as during the long pre-market characterization phases of drugs.

A? Further implementation of this innovation? linked to the culture and simultaneous analysis of single cells or cell aggregates over time. This aspect? of strong interest in the biomedical and clinical fields. For example, tracking changes in properties? physical samples during drug treatment? useful in the pharmacological field for applications such as personalized medicine, oncology and medically assisted procreation.

This implementation consists of a device and the respective method for measuring density? mass, weight, size and / or shape of a corpuscle for a desired period of time, up to several weeks. Also the method? non-destructive, and the specific corpuscle selected for the analysis can? be appropriately recovered after analysis for further investigation.

Although in the fluidic devices illustrated in Figures 2 and 3, the pumping systems 200 are linear, the pumping system 200 can? also include one or more? systems for the recirculation of liquid, and one or more? systems for the generation of a flow for the circulation of the liquid inside the pumping system 200 and the sedimentation chamber. In a specific implementation, represented in Figure 13, the pumping system 200 comprises a further recirculation channel 250 and a secondary pumping system, here called recirculation device 240 (Figure 13). The protocol for introducing the corpuscle into the fluidics device? similar to that previously described. A particle 420, contained in the analysis medium 450, is extracted from the reservoir 400 and introduced into the pumping system 200 through the inlet channel 210. The flow generation system 230 is activated to introduce the sedimentation chamber 100 into the corpuscle, which is monitored by the sensing device 300. The pumping system 200 further comprises a recirculation channel 250 circularly connected with the sedimentation chamber and a recirculation device 240 (Figure 13), which is activated by the processor 500 to maintain the corpuscle inside the sedimentation chamber 100, through the generation of a controlled circular flow on the basis of the particle data obtained from the detection system 300. The recirculation channel 250 allows the recycling of the analysis medium, with the advantage of reducing the expenditure of the same to a minimum.

An implementation of the embodiments of the present invention allows to measure the density? mass, weight, size and shape of the corpuscle before and after the replacement of the main analytical medium 450 with one or more? different analysis means 451. In this implementation the pumping system 200 further comprises one or more? secondary channels fluidically connected to the sedimentation chamber 100 for the introduction or removal of liquids and / or corpuscles from one or more? additional tanks. The secondary channels can be connected directly to the sedimentation chamber 100, or they can be connected through an inlet channel or through a secondary fluidic circuit. A specific implementation of this embodiment adopts a variation of the fluidic device described in Figure 2. The corpuscle 420 is introduced and kept suspended inside the first analysis medium 450, in the sedimentation chamber 100, through the pumping system 200. In this implementation, the pumping system 200 includes a secondary channel for buffer exchange 260 (Figure 14), connected to a second tank 401 which contains the new analysis medium 451. The system allows the introduction of the new analysis medium. analysis 451 inside the sedimentation chamber 100 through the secondary channel for buffer change 260. The corpuscle is kept inside the sedimentation chamber 100 during the replacement of the main analysis medium 450. This procedure, repeated a few times, leads to the complete exchange between the analysis means both in the sedimentation chamber 100 and in the recirculation channel 250. In a specific variant of this embodiment, the junction between the secondary buffer switching channel 260 and the input channel 210? a selection valve 270. In another variation of this embodiment, the system? adapted to draw from a tank 401 which contains the new analysis medium 451 through the same inlet channel 210 through which the corpuscle is introduced, by manual or automated replacement of the tank 400. In a further variation of this implementation, the pumping system 200 comprises the secondary channel for buffer exchange 260 (Figure 14), the recirculation channel 250, and the recirculation system 240 (Figure 13). This implementation allows to perform density measurements? mass, weight, size and / or shape of the corpuscle, during periods of time even several days or weeks, during which? It is possible to change the liquid in which the corpuscle is kept and analyzed. The combination of these modes? of use can? in fact, it can be advantageous for various applications. For example, the corpuscle can? be treated for a long period of time while frequent changes of the analytical medium are performed. What? doing, during each replacement? It is possible to introduce a second liquid that has characteristics different from the previous one, for example small variations in the concentration of a drug for the analysis of its toxicity. Otherwise, the treatment can? be more? intense, introducing a second means of analysis that has significantly different characteristics from the first, and then monitoring the reaction of the corpuscle to this variation. In an embodiment of the present invention, the same particle is analyzed when subjected to a sequence of analysis media with different ionic strength. Through this embodiment,? It is possible to measure the effect of the ionic strength on the corpuscle 420. In some ways of use, the use of the correct analysis medium? a critical aspect for the technical implementation of this embodiment: if the corpuscle has a semipermeable membrane (e.g. if it is a single cell or a spheroid), an isoosmotic test medium (e.g. PBS 0.9% w / v for cells) allows you to measure the density? of natural mass of the corpuscle. In a different embodiment of the present invention, the analytical medium is not? iso-osmotic with the corpuscle and the influence of the ionic strength on the corpuscle pu? be measured.

In a different embodiment of the present invention, the sequence of analysis means used to process the corpuscle 420 can be found. contain a drug, or other biologically active compound, which can influence the size, volume, density? mass and / or weight. In this case, the system measures the effect of the compound on the cell, spheroid or organoid. A potential result of density monitoring? mass and size of live samples,? shown in a graphical representation shown in Figure 15. Here, the corpuscle? considered having spherical geometry, and its size? shown in the graph as diameter. The example presented aims at recognizing the maturation stage of the spheroid, and the time necessary for its interaction with the drug. In this illustrative graph, the corpuscle grows in both diameter and density. mass, until the time T1 is reached. Then we observe the stabilization of the diameter, despite the density? keep increasing. This indicates a compaction phase up to time T2, when the density? reaches the plateau, in which the spheroid is in a condition of maturity? ideal for changing the analysis medium. The time T3 represents the moment relative to the introduction of a drug, and the subsequent trend shows its effect. This procedure offers the possibility? to observe and compare the variation of density? and diameter over time and to gather valuable details on the interaction of the corpuscle with the drug.

A variant of this embodiment relates to the combination of culture and long-term corpuscle analysis, with a solution for changing the analysis medium. In fact, a further important aspect of scientific research concerns the possibility? to monitor a sample during changes in its surrounding environment, such as pH, ionic strength, change in growth factors in the culture medium, or during specific treatments with pharmacological and / or chemical agents.

An additional modality? of use of the embodiment of the present invention allows the Sorting of corpuscles, ie the organization and sorting of subpopulations of corpuscles. This operation ? important in the biomedical field as it allows the collection of subpopulations of corpuscles based on specific properties? or geometric characteristics. Cellular Sorting? commonly used in medicine, pharmacology and scientific research in general. Its applications include, for example, the fields of regenerative medicine and personalized medicine, as well as those of anticancer therapy or virology. Current methods for cell sorting base the selection of samples on factors such as the presence of protein markers, or specific biochemical interactions. Currently, however, there is no technology capable of selecting and sorting a wide range of samples based on density. of mass, weight, size and shape. Therefore, another implementation of the present invention relates to a method, and relative device, for the selective sorting of the corpuscles based on the concomitant measurement of these properties.

In a specific embodiment of this implementation, the sedimentation chamber 100 includes a bifurcation that connects it to a recovery channel 120 in turn fluidically connected to a specific portion of the pumping system 200, here called secondary channel for Sorting 280 (Figure 16 ). Both the inlet channel 210 and the secondary channel for sorting 280 are controlled by a flow regulator, such as valves 270 and 290. In this implementation, a particle 420 is introduced into the sedimentation chamber 100 through the inlet channel 210 while the valve 290? closed and valve 270 is held open. Once the measurement of the particle is finished as described in the previous implementations, the results are evaluated for the Sorting of the sample based on the density values? of mass, weight, size and shape. The selected corpuscle is then collected through the activation of the pumping system 200, while the valve 270? closed and the valve 290 is kept open to allow the passage of the corpuscle through the recovery channel 120 and the secondary channel for Sorting 280, to finally reach the tank 700 (Figure 16).

In a variant of the present embodiment, the corpuscle is selected and ordered exclusively on the basis of its density? mass. This ? relevant for the decrease in inhomogeneity? of scientific tests, allowing for example the selection and collection of a subpopulation of spheroids having a similar degree of compaction. In another variant of the present embodiment,? possible to combine more? parameters measured for the selection of the collected corpuscles. For example, characteristics such as the standard deviation of the measured diameter and the velocity? terminal, can be used to sort corpuscles according to their degree of sphericity. This specific application could be relevant for testing the spread of drugs on different homogeneous batches of spheroids, and could provide information on the time it takes for the drug to reach the nucleus. This same application could also provide information on the concentration level of the drug required to efficiently cross a number of cell layers, as well as to carry out studies on its toxicity.

In further variations of the present embodiment, the parameters and limit values to be used for Sorting, could for example be defined by the user, or be extracted from databases; or they could be automatically defined by an unsupervised algorithm that can sort similar families of corpuscles within populations.

A further aspect of the innovation concerns the importance of translation from the manual to the automated process, particularly when moving from laboratory use to large-scale medical and industrial applications. The invention presented? conceived in such a way as to be easily automated through the use of existing devices, thanks to the low number of hardware components. These components can also be easily miniaturized with existing technologies.

Claims (13)

FLUID DEVICE FOR ANALYSIS OF BODIES AND RELATIVE METHOD CLAIMS 1. Fluidic device for the measurement of at least one of the densities? of mass and weight of a corpuscle, where said fluidic device includes: a sedimentation chamber fluidically connected to an inlet channel, where said inlet channel? configured to be immersed in a liquid; a pumping system connected to the sedimentation chamber, where said pumping system? suitable for controlling the flow of liquid in the sedimentation chamber; a processor configured to: obtaining particle data relating to a particle in at least one region of the settling chamber, e calculate at least one between density? mass and weight of the corpuscle based on the data received. The fluidics device according to claim 1, further comprising at least one sensing device configured to acquire the particle data and to provide the particle data to the processor. 3. The fluidics device according to claim 1 or 2, where the processor? configured to control the pumping system based on at least a portion of the received particle data. The fluidic device according to at least one of claims 1 to 3, where the particle data includes at least one of the velocity, shape, position, size of the corpuscle. The fluidic device according to at least one of claims 1 to 4, further comprising a temperature control device, configured to provide the processor with a temperature measurement of a liquid in the settling chamber; where the processor? configured to calculate at least one of the density? mass and the weight of the corpuscle also on the basis of said temperature measurement. 6. The fluidics device according to claim 5, where the temperature control device? also configured to regulate the temperature of the liquid in the sedimentation chamber based on at least one of the information provided by the processor and a predefined temperature value. 7. The fluidic device according to at least one of claims 1 to 6, further comprising a movable support suitable for housing at least a part of the at least one detection device, where the processor? further configured to guide the mobile support based on at least a part of the received particle data. The fluidic device according to at least one of claims 1 to 7, where the sedimentation chamber comprises a flow channel having a variable internal cross section, such as to allow the pumping system to induce flows in the sedimentation chamber having a non-zero horizontal component. The fluidic device according to at least one of claims 1 to 8, where the settling chamber comprises flow channels connected in parallel to each other and to the input channel, where each flow channel? also connected to a flow regulator, where the processor? configured for receiving input data relating to a particle in the input channel, and controlling the flow regulators on the basis of said input data. The fluidic device according to at least one of claims 1 to 9, where the pumping system comprises a recirculation channel connected in parallel to the sedimentation chamber, wherein said recirculation channel comprises a recirculation device, e where the processor? configured to control said recirculation device for the recirculation of the liquid in the sedimentation chamber. The fluidic device according to at least one of claims 1 to 10, where the pumping system further comprises a secondary channel fluidically connected to the sedimentation chamber, where the processor? configured to selectively control the flow in the secondary channel to inject liquid into the sedimentation chamber and / or to expel liquid from the sedimentation chamber through the secondary channel. 12. Method for the measurement of at least one of the densities? mass and weight of a particle, said method comprising: introducing a particle to be analyzed into a sedimentation chamber of a fluidic device through an inlet channel immersed in a liquid; obtaining particle data, where said particle data are relative to a particle in at least one region of the settling chamber, where said particle? moving in a liquid at rest in the settling chamber, e calculate at least one between density? mass and weight of the corpuscle based on the data received. The method according to claim 12, further comprising: select a corpuscle on the basis of one of the densities? of mass, weight, size and shape; And collect the selected corpuscle in a predefined container based on at least one of the densities mass, weight, size and shape of the corpuscle.